Extended Data Fig. 9: p97 unfolds and translocates along BgaA-T.
A-B. SDS PAGE of p97-Strep TagII (A) and BgaA-T (B) purified using Ni-NTA column from cell lysate of E. coli(DE3) cells expressing the proteins. Molecular weight of markers is mentioned in the side of the SDS gels. CFE: Cell-free extract; EF: Elution fraction. Protein expression was induced using IPTG (0.2 mM). C. Immunoblot showcasing the formation and binding of p97-NPLOC4-UFD1 complex on Strep-beads. D. Immunostaining of BgaA-T (a truncated version of BgaA, consisting of amino acids 1 to 1049) deposited on a hydrophilic coverslip using anti-(His)6 antibody. Scale bar, 20 µm. E. Beads (unconjugated or conjugated with p97) were trapped and brought close to the coverslip surface coated with BgaA-T or ubiqutinated BgaA-T. The percentage of beads that bound the surface was calculated and plotted for each of the conditions described in the figure. n > 20 beads. F. Distance from trap centre versus time plot the trap centre giving multiple force generating events (one of which is depicted in the green box). Representative beads conjugated with p97WT (Left) and p97E578Q (Right) are shown. Orange arrows indicate the time points at which piezo was moved. On moving the piezo, the p97E578Q mutant binds the surface but keeps falling back to the trap, however only a few beads escaped the trap. Once the piezo is moved, the p97WT bead binds the surface and extracts the protein. Hence the majority of the beads escape the trap (as shown). G. Streptavidin bead was moved in 20 nm steps in + and - Z positions and images were acquired at each step. A calibration curve – position versus intensity of greyscale histogram, was generated using the obtained values to calculate displacement for protein-conjugated beads, when they bind to BgaA surface. Blue dots represent intensity values obtained with only bead. Red triangles and green triangles are values obtained with p97WT and p97E578Q beads, respectively. H. Time-lapse images of representative p97WT and p97E578Q tagged bead translocating towards BgaA-T laden coverslip without moving the piezo stage. I. Graph representing the translocation kinetics of p97WT and p97E578Q beads towards the BgaA-T coated coverslip.
